W7-induced GPVI shedding is abrogated in Adam10^−/− platelets. (A) Washed platelets were treated with the indicated reagents (W7: 150μM, 1 hour at 37°C; CCCP: 100μM, 1 hour at 37°C), stained with a fluorophore-labeled antibody recognizing the indicated glycoproteins, and analyzed on a FACSCalibur. (B) Washed platelets were incubated with biotinylated JAQ1 and then treated with CCCP (100μM) or W7 (150 μM) for 1 hour at 37°C. Supernatants were applied on a JAQ3-coated ELISA plate, and GPVI-JAQ1^biotin complexes were detected with HRP-conjugated streptavidin. (C) Western blot detection of intact GPVI (JAQ1-HRP) in CCCP (100μM), W7 (150μM), or vehicle-treated platelets. GPIIIa was used as a loading control. Results of experiments in panels A and B are mean ± SD (n = 4 mice per group, representative for 3 individual experiments).